Vibrio cholerae is the causative agent of the devastating diarrheal disease cholera. This organism is also a natural inhabitant of aquatic environments worldwide. V. cholerae is thought to exist in these environments primarily in microbial communities called biofilms. Biofilm bacteria are more tolerant to environmental stresses such as UV radiation, desiccation, low pH, antimicrobials, and the immune defenses of the human host. This tolerance protects V. cholerae not only from the harsh conditions in the environment, but also from the acidic conditions of the host stomach. There is also evidence of V. cholerae biofilms in the host intestinal environment which suggests that V. cholerae might use biofilm formation as a long-term protective strategy in the human host. Development of biofilms is controlled by signaling networks that respond to chemical signals in the environment. Our long term goal is to identify and understand the signaling systems that regulate biofilm formation in V. cholerae. In this proposal, we focus our attention on a specific signaling system that responds to the polyamines norspermidine and spermidine. These polyamines act as environmental signals that are detected and processed by a signaling system composed of the proteins NspS and MbaA. NspS is the sensor protein of the system that detects these polyamines. MbaA is a phosphodiesterase that breaks down cyclic-diguanylate-monophosphate (c-di-GMP), one of the central modulators of biofilm formation in V. cholerae. We hypothesize that norspermidine and spermidine binding to NspS affects the interaction of NspS and MbaA which then results in changes in its c-di-GMP phosphodiesterase activity. This change is manifested as increases or decreases in c-di-GMP levels in the cell which lead to corresponding increases or decreases in biofilm formation. We have identified similar systems in diverse bacteria composed of NspS-like sensor proteins and MbaA-like transducer proteins which are predicted to respond to a variety of environmental clues including polyamines, phosphonate, phosphate and nitrate. The goal of the proposed project is to study the NspS/MbaA system further both to gain more insight into V. cholerae biofilm formation as well as a model for the NspS/MbaA-like c-di-GMP signaling systems. The specific aims of this project are to: i) further characterize the interaction by identifying a potential MbaA interaction surface on NspS through a mutagenic screen; ii) further characterize the enzymatic activity of MbaA and to understand the role of the GGDEF domain by mutational analysis followed by enzyme and biofilm assays; and iii) investigate the effect of norspermidine and spermidine on c-di-GMP synthesis using direct quantification, reporter-fusions assays, and an in vitro reconstitution assay.